While our past efforts have focused on defining the domains and motifs that constitute the building blocks for AKAP targeting of PKA, our focus now has shifted to understanding how large macromolecular signaling scaffolds are assembled. Our multi-scale approaches are allowing us not only to achieve a mechanistic understanding of such scaffolds at atomic level resolution but also to understand the cellular architecture of PKA scaffolds that are assembled at the mitochondria and how these scaffolds change in response to cAMP and as a function of diet and disease. We are focusing, in particular, on the uniqueness of the PKA isoforms as revealed by newly solved structures of full-length holoenzymes and on a newly discovered and highly conserved mitochondrial PKA substrate, ChChdS, that is localized to the intermembrane space. ChchdS nucleates a large scaffold that bridges the inner and outer membranes and plays a major role in crista biogenesis [1]. Also recruited to this complex is a novel Rl-specific AKAP, Sphingosine Kinase Interacting Protein (SKIP), recently discovered by Scott and Taylor [2, 3]. Depletion of ChchdS leads to loss of crista and loss of oxidative metabolism while ChchdS is enriched in livers of mice fed on a high fat diet (HFD), which is associated with insulin resistance and obesity, hallmark symptoms of Type II diabetes. We will use the expertise of the Analytical Core (Core A) to define the molecular features of these complexes while the Multi-Scale Ceil Imaging Core (Core B) will be used to define the architecture of the mitochondria and how it changes dynamically in response to cAMP and to diet and depletion of ChchdS. In parallel, we are using miniSOG (small Singlet Oxygen Generator) techno-logy, recently discovered by Tsien and Ellisman [4] to finely map localized sites of PKA signaling. MinSOG, a genetically encoded tag that generates singlet oxygen that can then be used for EM contrast, has the potential to do for EM what GFP did for fluorescence microscopy. Moving from atomic level resolution to defining the dynamic molecular architecture of organelles is the driving motivation for Protect I as we try to understand how specificity in PKA signaling is achieved through dynamic and polyvalent signaling scaffolds. Our specific goals are as follows. In Aim I we will crystallize an Rl-specific AKAP bound to full-length Rip, an isoform that is enriched in mitochondria. In Aim II we define the molecular and dynamic features of ChChdS using peptide arrays, structural biology, and mass spectrometry, in Aim III we use miniSOG reporters to define PKA targeting sites and to correlate PKA signaling with mitochondria dynamics. In Aim IV we will collaborate with Olefsky to elucidate tissue-specific changes in PKA signaling and mitochondria morphology in response to a HFD. RELEVANCE: Although PKA plays a major role in regulating metabolism and mitochondrial dynamics, we lack knowledge of how macromolecular signaling scaffolds target specific PKA isoforms to mitochondria and how they are regulated. The multi scale strategy outlined here will define structural and cellular PKA scaffolds at the mitochondria and elucidate how these scaffolds and mitochondrial dynamics change in response to diet induced insulin resistance and obesity, hallmarks of type II diabetes. Novel targets for intervention will likely emerge as well as enhanced molecular understanding of biological signaling by PKA.